PROJECT SUMMARY Substance use disorders (SUDs) are among society's most pressing challenges. Despite their high prevalence, effective interventions for SUDs are lacking. Considering the chronically relapsing nature of SUDs, understanding factors that contribute to relapse risk is a particularly important research goal. One such factor is biological sex. Once a SUD is established, women experience greater difficulty abstaining and relapse incidence, and resume use for longer periods of time after relapse has occurred when compared with men. Sex differences in relapse vulnerability are mediated in part by sex hormones, including estrogens. We have identified a novel mechanism through which estrogen acutely promotes drug seeking via actions in the prelimbic prefrontal cortex (PrL-PFC). Estradiol (E2), at a dose that produces proestrus levels in female rats, potentiates cocaine-primed reinstatement in females via estrogen receptor beta (ER?) activation in the PrL-PFC. Our preliminary findings suggest that this effect of E2 is associated with an ER?-dependent attenuation of inhibitory synaptic transmission in the PrL-PFC that is likely attributable to reduced GABA release. This proposal will build on these findings by further investigating the PrL-PFC mechanisms through which E2 regulates synaptic transmission and drug- seeking behavior (Aim 1) and by characterizing the PrL-PFC output pathways that are regulated by E2 and contribute to drug seeking (Aim 2) with a focus on projections to the nucleus accumbens (NAc) core and paraventricular thalamus (PVT). Women are particularly susceptible to relapse during periods of stress. Consistent with this observation, we have established a preclinical approach for examining the ability of stress to potentiate cocaine seeking and have demonstrated that, in female rats, stress further increases cocaine- primed reinstatement, thus producing an increased cumulative risk for drug seeking in females relative to males. We have found that stress-potentiated cocaine seeking is mediated by the stress hormone corticosterone (CORT) and that, like E2, involves CORT actions in the PrL-PFC that likely include attenuated synaptic GABA release and reduced constraint of PrL-PFC output pathways (i.e., to the NAc core) that are critical for drug seeking. However, while CORT-potentiated drug seeking involves endocannabinoid/CB1 receptor-dependent reductions in GABA release from cholecystokinin+ interneurons in the PrL-PFC, the actions of E2 are CB1R- independent and likely involve ER? regulation of fast-spiking parvalbumin+ interneurons. We will test this hypothesis and further explore the interactive relationship between CORT and E2 in Aim 3. This innovative multi- PI proposal pulls together expertise in synaptic physiology, systems neuroscience, and behavior analysis. The aims have broader implications for understanding how hormonally mediated reproductive and stress ?brain states? converge in the prefrontal cortex to guide behavior.